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1.
The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.Introducing DNA-modifying enzymes rather than DNA-based expression cassettes is an attractive alternative for genetic engineering and genome-editing applications such as gene targeting or site-specific recombination. It offers a transient presence of the enzymes, and the process can be coordinated with high levels of enzymatic activity at the time and sites of the desired DNA recombination events. Many DNA-metabolizing enzymes (endonucleases, transposases, and topoisomerases), when delivered in an unrestrained manner, show adverse effects on cell viability. Delivery in the form of protein or RNA may help to mitigate these effects (Cui et al., 2011; Sander et al., 2011; Watanabe et al., 2012). In addition, by introducing proteins, one can avoid the need to remove the protein-encoding DNA fragments from the engineered plant genome. This may help shorten the time from laboratory to field for future improved germplasms.Site-specific recombinases such as Cre or FLP have been widely used in genetic engineering applications (Sorrell and Kolb, 2005). The 38-kD Cre enzyme specifically binds to and recombines the 34-bp loxP sequences, allowing the removal, integration, or inversion of the DNA fragment flanked by these sequences (for review, see Wang et al., 2011). There are a number of established methodologies designed to provide the Cre recombinase activity for site-specific recombination in eukaryotic cells that do not involve the delivery of DNA. These methods include lipofection (Baubonis and Sauer, 1993), microinjection of protein or mRNA (de Wit et al., 1998; Luckow et al., 2009), electroporation of protein or mRNA (Kolb and Siddell, 1996; Ponsaerts et al., 2004), or using modified microorganisms for Cre delivery to their host cells (Vergunst et al., 2000; Koshy et al., 2010). Another strategy that has been used is the incubation or injection of tissues/cell cultures with cell-permeant Cre, a modified Cre protein fused to protein transduction domains or cell-penetrating peptides (Jo et al., 2001; Will et al., 2002; Lin et al., 2004; Nolden et al., 2006).For biotechnological applications in plant sciences, protein delivery systems have been developed, including microinjection (Wymer et al., 2001), protein immobilization to gold particles (Wu et al., 2011), and protein transduction through cell-penetrating peptides (for review, see Chugh et al., 2010). The cell-penetrating peptides were shown to enable intracellular delivery of the Cre recombinase protein to rice (Oryza sativa) callus tissues (Cao et al., 2006). Nanobiotechnology is offering an attractive alternative, since nanoparticles can be precisely tailored to deliver a particular biomolecule to the cell, tissue, or organism of interest when needed (for review, see Du et al., 2012). Mesoporous silica nanoparticles (MSNs) are particularly suited for this purpose. These porous nanoparticles are formed by a matrix of well-ordered pores that confers high loading capacity of molecules like proteins (for review, see Popat et al., 2011). Additionally, surfaces of MSNs can be readily modified, permitting the customization of nanoparticles to particular experimental needs (for review, see Trewyn et al., 2007). In our previous studies, it was shown that MSNs can be used for the codelivery of DNA and chemicals (Torney et al., 2007) as well as DNA and proteins (Martin-Ortigosa et al., 2012a) to plant cells via biolistics. To improve MSN performance as a projectile, gold plating of MSN surfaces was performed, increasing nanoparticle density and, subsequently, the ability to pass through the plant cell wall upon bombardment (Martin-Ortigosa et al., 2012b).In this work, the Cre recombinase enzyme was loaded into the pores of gold-plated MSNs and delivered through the biolistic method to maize (Zea mays) cells containing loxP sites integrated into chromosomal DNA (Lox-corn; Fig. 1A). Lox-corn expressed the glyphosate acetyltransferase gene (gat) and the Anemonia majano cyan fluorescent protein gene (AmCyan1) flanked by loxP sites. The MSN-released Cre enzyme recombined the loxP sites, thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of a variant of Discosoma sp. red fluorescent protein gene (DsRed2) and the loss of the selectable marker gene (Fig. 1A). Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues, leading to successful genome editing.Open in a separate windowFigure 1.A, Schematic representation of the MSN-based bombardment technology. Cre protein is loaded into the pores of gold-plated MSN (Cre-6x-MSN) and subsequently bombarded onto immature embryos of a transgenic maize line carrying a loxP construct (Lox-corn). The parental transgenic Lox-corn tissues are blue fluorescence and herbicide resistant because they harbor a cassette with the glyphosate acetyltransferase (gat) selection gene and the AmCyan1 (cyan) marker gene flanked by the loxP sites. The DsRed2 (dsred) gene for the expression of a red fluorescent protein is placed downstream of the cassette. Once Cre recombinase is released inside the cell, it performs the recombination, excising gat-AmCyan1 genes and leading to the expression of the DsRed2 gene, switching the cell fluorescence pattern from blue to red. P, Promoter; T, terminator. UBINTRF, CYANF, and DSRED2R are primers for DNA analysis. B, Transmission electron microscope image showing the typical hexagonal shape and the well-ordered pore structure of a 6x-MSN. C, Scanning electron microscope image showing gold nanoparticle deposition (white dots) in all surfaces of 6x-MSN. D, Western blot showing Cre protein loading and release dynamics from 6x-MSN. The protein loading is almost immediate, even though some protein can be detected in the buffer even after 1 h of loading. For the release, some Cre protein can be observed after 24 h of incubation. Most of the protein remains in the 6x-MSN pellet. C+, 400 ng of Cre protein; Empty, a lane with no protein loading. The bands observed in the Empty lane were the spillover from the neighboring Pellet lane, which represents Cre-loaded 6x-MSN after the release experiment resuspended in Laemmli loading buffer (see “Materials and Methods”).  相似文献   
2.
张九东  王琳  隋洁  田先华  任毅 《植物学报》2018,53(2):212-218
毛茛科类叶升麻(Actaea asiatica)有2种开花方式, 一种与同属其它植物相同, 花萼片在开花早期脱落, 花药远离柱头; 而另一种较为特殊, 能使该植物进行自花授粉, 即在花开放前, 花萼从花托上脱离, 但萼片并不张开, 部分雄蕊的花丝伸长, 将花药推入萼片和柱头之间, 收缩的花萼片将开裂的花药压在宽大的柱头上, 进而完成自花授粉。套袋实验结果表明, 类叶升麻自发自交具有很高的结籽率, 不具有孤雌生殖和风媒传粉。繁育系统估测分析结果显示, 类叶升麻自交亲和,为兼性自交的繁育系统。  相似文献   
3.
Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.  相似文献   
4.
5.
A Bacillus-like isolate, strain FJAT-13831T, isolated from the No. 1 pit soil of Emperor Qin’s Terra-cotta Warriors in Xi’an City, China, was studied to determine its taxonomic status. Dominant fatty acids of this organism included iso-C15:0, iso-C17:0, C16:0, iso-C13:0, anteiso-C15:0, and iso-C17:1ω5c. Comparative 16S rRNA gene sequence analysis confirmed the affiliation of this isolate to the genus Bacillus and indicated that it was closely related to Bacillus pseudomycoides DSM 12442T (99.72 % similarity). A phylogenetic analysis of the gyrB gene sequence similarities exhibited independent clustering of the isolate FJAT-13831T and showed 93.8 % (<95 %) sequence similarity with its closest phylogenetic neighbour B. pseudomycoides DSM 12442T. Separate standing of the strain FJAT-13831T was supported by a whole genome-based phylogenetic analysis with an average nucleotide identity value of 91.47 (<95 %) between isolate FJAT-13831T and B. pseudomycoides DSM 12442T and was consistent with the results of DNA–DNA hybridization (69.1 % relatedness). These findings support the conclusion that the isolate FJAT-13831T represents a novel species, for which the name Bacillus bingmayongensis sp. nov. is proposed. The type strain is FJAT-13831T (= CGMCC 1.12043T = DSM 25427T).  相似文献   
6.
摘要:【目的】筛选可产生抗血栓活性物质的细菌。【方法】利用VY/4平板、酪蛋白平板从水样、土样、兔粪、羊粪、朽木等20多个样品中筛选目的菌株;利用纤维蛋白平板和纤维蛋白试管检测抗血栓活性;利用形态学特征、理化性质、16S rRNA序列同源性鉴定目的菌株。【结果】得到5株可产生抗血栓活性物质的细菌,重点研究了菌株LDS33,发现其分泌的胞外蛋白在纤维蛋白平板上和纤维蛋白试管中均显示出强烈的溶栓活性,通过试管法发现此蛋白质同时具有较强的抗凝活性。结合形态学、理化性质、16S rDNA序列及进化树分析,发现该菌株属于硬壁菌门芽孢杆菌目芽孢杆菌科芽孢杆菌属的短小芽孢杆菌,将其命名为Bacillus pumilus LDS.33。【结论】短小芽孢杆菌LDS33可产生高活性的抗凝溶栓双活性蛋白。  相似文献   
7.
The drug–serum albumin interaction plays a dominant role in drug efficacy and disposition. The glycation of serum albumin that occurs during diabetes may affect its drug‐binding properties in vivo. In order to evaluate the interactivity characteristics of cyanidin‐3‐O‐glucoside (C3G) with human serum albumin (HSA) and glycated human serum albumin (gHSA), this study was undertaken using multiple spectroscopic techniques and molecular modeling analysis. Time‐resolved fluorescence and the thermodynamic parameters indicated that the quenching mechanism was static quenching, and hydrogen bonding and Van der Waals force were the main forces. The protein fluorescence could be quenched by C3G, whereas the polarity of the fluorophore was not obviously changed. C3G significantly altered the secondary structure of the proteins. Furthermore, the interaction force that existed in the HSA–C3G system was greater than that in the gHSA–C3G system. Fluorescence excitation emission matrix spectra, red edge excitation shift, Fourier transform infrared spectroscopy and circular dichroism spectra provided further evidence that glycation could inhibit the binding between C3G and proteins. In addition, molecular modeling analysis supported the experimental results. The results provided more details for the application of C3G in the treatment of diabetes.  相似文献   
8.
目的 探讨甘露聚糖结合凝集素(Mannan-binding lectin,MBL)对CD11c+髓样树突状细胞(CD11c+mDC)表型和功能的影响.方法 应用磁珠分选技术获得BALB/c小鼠脾脏CD1 1c+ mDC和CD4+T淋巴细胞.在CD11c+mDC中加入不同浓度的MBL(2.5~20 μg/mL)刺激,以不加MBL的细胞作为对照,应用ELISA法检测细胞培养上清液中的IL-12水平,流式细胞仪检测细胞表面分子CD40、CD80、CD86及HLA-DR的表达.用MTT法测定CD11c+mDC刺激CD4+T淋巴细胞的增殖能力.ELISA法检测细胞培养液中IL-4和IFN-γ水平.结果 MBL显著增强CD11c+mDC表面分子CD40、CD80、CD86及HLA-DR的表达和IL-12的分泌,促进CD4+T淋巴细胞的增殖和抗原递呈能力,诱导CD4+T向TH1反应分化.结论 MBL能够有效刺激CD11c+mDC的活化,诱导CD4+T淋巴细胞向TH1反应分化.  相似文献   
9.
本文以鼻咽癌细胞株CNE2为放射敏感性的研究对象,经不同剂量X射线照射及不同时间培养后分别提取总蛋白,用共聚焦显微拉曼光谱仪检测其拉曼光谱。统计分析表明:被测样品的拉曼光谱中观察到一些可以归属于蛋白质物质的较为明显的基团频率振动峰;不同剂量的X射线照射后,总蛋白质的平均拉曼光谱与对照组谱形基本一致,但与对照组间的光谱存在着对应峰信号强度的不同。实验提示:照射后谱峰强度的增大或减小,提示着相关物质含量有所改变。分析照射后癌细胞总蛋白拉曼光谱的变化情况,结合数学统计方法,以探寻放射敏感性的特征拉曼标志,可以作为研究肿瘤放射敏感性的手段之一。  相似文献   
10.
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